Project 2, headed by Dr. Derek Abbott, will will test the alternative hypothesis that exaggerated N0D2 signaling leads to chronic intestinal inflammation. This project will investigate the dysregulation of the Nod2 gene at the molecular level. Lack of coordination between inflammatory signaling pathways influences the development of CD. We recently published that the E3 ubiquifin ligase ITCH, causes K63-linked polyubiquitinafion of RIP2, and this event downregulates active NOD2:RIP2 complexes. Mice in which Itch is genetically lost (itchy mice) develop inflammatory disease at mucosal surfaces (including intestinal inflammation). We have data showing that ITCH-/- mice develop gastritis, ileitis and colitis and that drugs that inhibit RIP2 tyrosine phosphorylation, such as tarceva and iressa, inhibit the exaggerated N0D2 responses. The central hypothesis of this project is that ITCH downregulates NOD2;RIP2-induced NFDB signaling, and that CD results when this downregulation is lost. Aim 1 will study this ITCH-induced ubiquitination event to determine the biochemistry and physiologic function of ITCH-induced RIP2 ubiquitination. In Aim 2, we will determine the role of tyrosine phosphorylation of R1P2 and the role that pharmacological inhibition of this phosphorylation plays in ITCH-induced R1P2 ubiquitination and N0D2- induced cytokine responses. In Aim 3, we will characterize the Gl inflammation of the Itchy mouse and will determine whether tarceva or iressa can alleviate chronic intestinal inflammation in these mice. Our preliminary data shows that Itchy mice have increased gastrointestinal permeability. This increased permeability allows MDP leakage into the lamina propria and causes prolonged NFkappaB activation to occur. We hypothesize that tarceva and iressa will inhibit exaggerated lamina propria N0D2 activation in the itchy mice and alleviate the chronic inflammation. The overall objective of this project is to determine the biochemistry of the ITCH:RIP2 interaction, the physiologic significance of this interaction, and most importantly, whether this interaction can be inhibited pharmacologically to ameliorate chronic intestinal inflammation. RELEVANCE (See instructions): CD affects more than 500,000 individuals in the US and incurs significant costs to society. Understanding the precise mechanisms and immune defects that cause the disease will allow us to develop better therapies and begin to develop a cure for this devastating disease.